Hybrid Advances 2 (2023) 100023

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Hybrid Advances
journal homepage: www.journals.elsevier.com/hybrid-advances

Review Article

Recent advances in electrochemical biosensors – A brief review

M.S. Sumitha, T.S. Xavier *
Centre for Advanced Materials Research, Department of Physics, Government College for Women, Thiruvananthapuram, University of Kerala, 695014, India

A R T I C L E I N F O A B S T R A C T

Keywords: A sensitive biological system and a detector system with appropriate transducers for obtaining the output signals
Electrochemical biosensor classification make up a biosensor. These devices have a wide range of uses, including disease screening, the detection of
Amperometric biosensors environmental pollutants, agriculture, and routine medical examinations. The product's selectivity, sensitivity,
Potentiometric biosensors
stability, and lower production costs will all be critical factors in its widespread commercialisation. Recently,
Impedimetric biosensors
Voltammetric biosensors
scientists have tackled the issue of developing a nano biosensor with a high degree of sensitivity and selectivity for
the recognition of biomarkers of immune responses and cancer. The fact that electrochemical nano biosensors
may successfully be employed for detecting medications in addition to pathogen biomarkers has significantly
altered the COVID picture in our favour. During the worldwide epidemic, a number of cutting-edge SARS - CoV-2
biosensors with portable, smartphone-connected instant detection devices were helpful. This article provides a
concise summary of the underlying working principle, generations of developments and numerous detection
methods employed by electrochemical biosensors. The difficulties, knowledge gaps, and possible solutions in the
field of electrochemical biosensors are all discussed in the context of this categorisation. This novel overview also
sheds light on the different kinds of electrochemical biosensors and the tasks they perform, as well as recent
developments in the “smart biosensor” industry with potential future directions and challenges for this growing
subject.

1. Introduction nano biosensor's performance heavily depends not only on the surface
science of the nanomaterials but also on surface engineering and
A biosensor is a self-contained, unified device that uses biological functionalisation.
materials in direct spatial connection with a transduction component to The recognition of an electric signal created by an electro-active
explore an analyte. The trio-combo design of a sensitive biological system species, either generated or drained by an enzymatic reaction, is the
and the detector system with appropriate transducers for obtaining the foundation of electrochemical biosensors, particularly the enzyme-
output signals have a wide range of uses, including disease screening, the coupled ones. Ezymes offer excellent selectivity and catalytic activity,
detection of environmental pollutants, agriculture, and routine medical but they will eventually lose that activity, which will limit the life of the
examinations. The monitoring of industrial oxygen in the 1950s by biosensor. A voltage is provided to the electrode when the enzyme layer
Leland C. Clark marked the beginning of the application of electro- catalyses the synthesis or depletion of an electro-active species, which
chemical sensors. A minimum of two electrodes—the sensing and drives the redox reaction and produces a signal. The concentration of the
counter electrodes—must be present for an electrochemical sensor to analyte is correlated with this electrical signal. Due to their excellent
function. These electrodes must be in touch with one another via either selectivity and simplicity, electrodes are frequently used as signal
an external electric circuit or an electrolyte. The primary medical purpose transducers. Additional selectivity is provided by electrochemical
of oxygen sensor design consists of two electrodes in a cell with an ox- detection since various electro-active compounds can be oxidised or
ygen permeable membrane separating them from the electrolyte solu- reduced at various potentials. Additionally, electrochemical detection
tion. Through membrane diffusion, oxygen was lowered in the indicator can be used with current downsizing, and microfabrication techniques
electrode. This led to a current that was inversely correlated with the require little power and are not affected by sample turbidity or nature.
oxygen content of the sample [1]. Fig. 1 illustrates the general working The majority of enzyme-based electrochemical biosensors are quite
principle behind electrochemical biosensors [2]. The electrochemical affordable because they don't need a lot of equipment. Enzyme electrodes

* Corresponding author.
E-mail addresses: sumithamnair@gmail.com (M.S. Sumitha), xavierkattukulam@gmail.com, xavier@gcwtvm.ac.in (T.S. Xavier).

https://doi.org/10.1016/j.hybadv.2023.100023
Received 5 October 2022; Received in revised form 12 January 2023; Accepted 12 January 2023
Available online 25 January 2023
2773-207X/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

The partial solubility of O2 is another limiting reagent, causing sensor

response alterations and oxygen deficit at greater glucose concen-
trations, leading to smaller glucose readings [5].

Hydrogen peroxide oxidation at an electrode surface requires an
extremely high voltage. Blood sample matrices contain electro-active
compounds that, upon redox, are known to induce interference. Since
other electro-active species introduce noise, the S/N (signal-to-noise
ratio) and detection limits (DLs) suffer [5].

With selectively permeable membranes like cellulose acetate or

Nafion placed between the sample and the enzyme-decorated electrode,
the interfering disturbance from electro-active species has been greatly
Fig. 1. Schematic illustration of the working principle of an electrochemical reduced, allowing for a more robust electrochemical response.
biosensor [2].
1.2. Second generation
are used in many clinical and point-of-care settings for analysing a wide
range of analytes. This slow electron transport, in this generation of electrochemical
Transduction has been simplified and improved, as evidenced by the biosensors, is due to the fact that the FAD reaction site of GOx is hidden
development of electrochemical biosensors. The benefits of nanomaterial by a protein layer [6]. Because of this, transferring electrons directly from
alteration have been increasingly investigated in recent years. Incorpo- one source to another is impossible without intermediaries. The oxida-
rating nanoparticles into the production process has dramatically tion mediator regenerates the FAD while simultaneously self-reducing in
increased the sensitivity and enhanced success and specificity of bio- the second-generation biosensor design. As the reduction mediator is
sensors. Especially with the use of nanomaterials and the Internet of replenished at the electrode surface, an electric signal is produced.
Things, the sensor sector has been revolutionised by portable, econom- Desirable mediators are nontoxic, chemically stable in reduced and oxi-
ical, tiny, convenient, wearable, flexible, implantable, transparent, dised forms, water-soluble, and have a relatively low detection potential.
lightweight, multifunctional, biocompatible, and quick sensing devices. Dissolving the mediator in the electrolyte speeds its transit from the
Intelligent, real-time monitoring of a wide variety of environmental electrode to the active site of the enzyme. PVlm [poly (vinylimidazole)]
stimuli requires Bluetooth, the internet network, and compatibility be- and PVP [poly(vinylpyridine)]-the mediators connected to
tween the biosensing array and these. These sensors are extremely robust osmium-complex electron relays, brought polymer redox sites and
and can survive harsh conditions. The quality of medical care is improved enzyme FAD redox arms together [6]. By using a synthetic mediator—a
by the IoMT (Internet of Medical Things). Achieving the desired level of to and fro electron transport molecule—instead of dissolved O2 in syn-
sensitivity and miniaturisation depends heavily on nanotechnology and thesising H2O2, 2nd generation biosensors fixed many of the problems
an in-depth understanding of AI. Closing the gap between nanotech- with the first generation [5].
nology and AI-assisted systems interfaced with IoMT technology con-
nected to Cloud services is crucial for the development of cutting-edge 1.3. Third generation
healthcare solutions like nanorobotics, nanomedicine, wearable contin-
uous real-time monitoring devices, etc. By placing the enzyme and intermediary together on the surface of
The generational development of electrochemical biosensors, thus, the electrode or in a neighbouring matrix, third-generation biosensors
depends on the chronological advances in material science and tech- effectively “wire” the enzyme to the electrode. Delivering electrons from
nology. Using a more generalised concept of analyte for the most recent the active site of the enzyme to the electrode, these immobilised medi-
developments, the first half of this article presents a brief summary of the ators function as non-diffusion redox control centres. In some circum-
evolution of electrochemical biosensors from the first to the fifth gen- stances, enzyme-to-electrode direct electrical contact increases electron
eration. The second half of this article examines how electrochemical transfer efficiency. Immobilised mediators facilitate effective electron
biosensors have developed in response to different electrochemical transport in 3rd generation biosensors, increasing current density. Fewer
transduction measuring modalities. reaction times are achieved by placing enzymes and mediators close to
the transducer surface, where the distance between them is shorter, and
an electron can more easily move from one to the other. The biosensor
1.1. First generation film prevents mediators from escaping into the surrounding environ-
ment, enabling in vivo monitoring. Applying the desired voltage to the
First-generation glucose biosensors used trapped glucose oxidase, electrode eliminates background interference. No reagents are needed;
GOx, to oxidise β-D-glucose to β-D-gluconolactone while simultaneously therefore, repeated and prolonged experiments are possible [7].
reducing FAD (flavin adenine dinucleotide) to FADH2 (two electrons, two It can be seen as an attempt to build an effective and specific trans-
protons reduction process result of FAD). After that, dissolved O2 would duction route that has been reflected in the development of the species
be used to create H2O2 (hydrogen peroxide), which would then be used over many generations [7]. Several efficient methods have been devel-
to renew the FAD from FADH2. Then the application of the voltage would oped to bring the enzyme and transducer close together without altering
oxidise the H2O2 on the electrode surface, generating an electric signal. the enzyme's structure or decreasing substrate accessibility to the active
Weaknesses in the design of first-generation biosensors are listed out core. There hasn't been much of an improvement in the transducer's base
below. characteristics due to these developments. The selectivity, S/N ratio, and
detection limit of a biosensor may all be improved with transducer

Active enzyme site and FAD functional group reside deeply within the tweaks [7].
molecule, making the reagents tough to reach.

In addition, according to the Marcus hypothesis, the rate of electron 1.4. Fourth generation
transport decreases exponentially with distance [3]. Enzyme “hot
spots” are often hidden deep into the protein core [4]. The electrons' The field of electrochemical biosensor fabrication has seen significant
“migration” from the enzyme and subsequent arrival at the electrode success from the use of electrodes modified with nanomaterial func-
interface is thus impeded. tionalisation in recent years. In their most basic form, sensors are

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M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

Fig. 2. Schematic reproduction of the steps involved in the chemiresistor fabrication.

composed of sensor scaffolds, sensing material, and electrical circuitry lower atomic number transition metal element and “X” might be nitro-
[8,9]. NH3 is present in exhaled human breath and works as an important gen, carbon, or both. The MAX phase is characterised by its layered
biomarker for detecting stomach lacerations or ulcers, renal disease due hexagonal structure and a stoichiometry defined by the expression Mnþ1
to H. pylori [Helicobacter pylori] infection, and, most recently, COVID-19 A Xn (n ¼ 1, 2, or 3). The universal formula for MXene looks like this:
[10,11]. Urea accumulates in the blood as ammonium ions due to renal Mnþ1 Xn Tx, where (nþ1) represents the number of levels of layers that
failure, and these ions can cross the lung membrane and cause damage. make up “X,” and “T” refers to the various terminals on the material's
People with peptic lesion disease and hepatic impairment have elevated surface [9]. It is possible to synthesise hybrid MXene-polymer nano-
levels of NH3 in their exhaled breath, with concentrations ranging from composites by either an ex-situ or an in-situ synthesis approach. Finally,
0.82 to 14.7 ppm [12]. To save lives, protect the environment, and the detecting surface of the hybrid MXene-polymer nanocomposites is
advance medical research, NH3 detection, even at minuscule levels, has deposited on a suitable substrate, typically featuring conducting elec-
therefore become a pressing concern. In a recent study, Chaudhary et al. trodes such as interdigitated and parallel electrodes, to complete the
provide a thorough explanation of how the next-generation chemiresistor chemiresistor's architecture. Spin coating, dip coating, electrospinning,
works with MXenes, shorthand for modified and functionalised hybrid inkjet printing, drop casting, spray coating, etc., are only a few of the
metal carbides/nitrides composites [13]. Nano-dimensional hybrids of many deposition processes available [9,14–16]. In Fig. 2, the procedures
polymer and MXene, exhibit novel physical and chemical properties due for making a chemiresistor are reconstructed from Ref. [9].
to the multi-interactions between their constituent molecules. In the The nature of the surface termination has a substantial influence on
context of 2-dimensional inorganic materials, MXene is a novel class that how the electrons transfer occurs, and are shown by the equations [9]
can be created by selectively removing elements from the “A”; the 13 or below.
14 group element from the precursor MAX phase, where “M” stands for a
2 NH3 þ3 O ¼ N2 þ 3 H2O þ 3e (1)

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M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

Fig. 3. Schematic illustration of the AI-IoMT assisted fifth generation biosensors.

“Internet of Things” (IoT) refers to a network in which everyday objects

NH3 þ OH ¼ NH2þ H2O þ 3e (2) are embedded with sensors, processors, software, and other technologies
that allow them to communicate and share information with other sys-
A rise in the impedance of the p-type MXene material is brought on by tems and platforms. IoT creates a vast, omnipresent network that enables
this phenomenon, which generates electrons and triggers electron-hole the integration of people, machines, and things at any time and in any
recombination. A drop in the MXene sample's resistance happens if it location by fusing various information-detecting devices with the
has an n-type nature. The sensing technique for particular air pollutants is internet; which can be widely used in the medical field for real-time
thus determined by the electrical characteristics of the MXene. The monitoring, data collection and analysis for research, of the hospital-
sensing characteristics of hybrid MXene-polymer nanocomposites are ised or non-hospitalised patients [19]. Hence the Internet of Medical
determined by the nature of the newly generated hetero interfaces, such Things (IoMT) has been referred to as “Smart Healthcare” [18], and it is
as (p-p) type or (p-n) type junctions amongst the precursors, and the the technology that will integrate the many healthcare resources and
weak chemical interactions [9]. services to create a digitalised healthcare system. The main advantage of
Precursor processing technologies, stoichiometric optimisations, and this combined Artificial Intelligence (AI) and IoMT-based sensor system
device fabrication all need significant improvement if the hybrid MXene is that it allows us the remote health tracking and emergency alert sys-
– polymer nanocomposites based chemiresistors are to be used for the tems. These health monitoring instruments can range from the straight-
biomarker detection. Fabrication strategies, precursor optimisation, forward monitoring of heartbeat, blood pressure, and blood sugar rate to
attaining improved sensing characteristics, scalable mass manufacturing, the more complex monitoring of specialised implants such as pace-
minimising environmental adulteration, and lowering nano-based/ makers, Fitbit wrist track bands, or cutting-edge hearing aids [18].
related waste production are among the critical hurdles for their prac- Some hospitals have begun using “smart beds,” equipped with motion
tical uses. Problems with high-performance chemiresistors’ commercial sensors that detect a patient's movements by measuring the mechanical
manufacture at a reasonable cost are another concern [9]. property changes caused by the patient's motion. Fig. 3 shows the sche-
matic diagram of the various fields in which the fifth-generation bio-
1.5. Fifth generation sensors, in general, find applications in the medical field. Fig. 4 shows a
simplified timeline of glucose sensor evolution, across generations.
New technologies are desperately needed for bioanalyte live moni-
toring, autonomous control, and exact detection. With the advent and 2. Electrochemical biosensor classification
widespread use of ideas like big health, the Internet of Things (IoT), and
big data, biosensing has ushered in a new era [17]. Wearable biosensors Increased sensitivity and efficiency have resulted from the develop-
are being developed to provide individuals with more knowledge of their ment of nanotechnology in the biosensor sector. Nanomaterials, with
own physiological dynamics, thereby overcoming obstacles associated their high surface-to-volume ratio, are responsible for this improvement.
with centralised, reactive healthcare systems. However, the output Almost all existing biosensors make use of some form of electrochemical
analysis becomes extremely challenging due to the intricate causation detections, such as amperometric, voltammetric, or impedimetric de-
involved. By combining artificial intelligence (AI) techniques like pattern tections or the detection of deoxyribose nucleic acid (DNA) by chemical
recognition and algorithms with biosensors, we can close the loop be- processes such as hybridisation [20]. The working, reference and counter
tween data collection and processing, leading to more precise diagnoses electrodes make up electrochemical biosensors. Enzymatic catalysis re-
and treatments. sults in the production or consumption of an electron, which is then
By combining cutting-edge machine learning algorithms with wire- employed to provide a signal that aids detection [21]. Detailed expla-
less biosensing technology, artificial intelligence-powered biosensors nations of the various ways in which electrochemical biosensors might be
show significant promise for enabling continuous healthcare monitoring categorised are discussed below.
and cloud-connected Point of Care (POC) diagnostics [17]. The term

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M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

Fig. 4. Development of electrochemical glucose biosensor.

2.1. Amperometric μA mM-1cm2 [23]. The efficiency of these techniques and parameters
influences the range of glucose concentrations that can be detected
Current generated by an oxidation reaction can create an ampero- properly.
metric biosensor, a device capable of providing precise quantitative The cyanobacterium strain Anabaena variabilis was used to develop
analytical data. In most cases, these biosensors can compete with an amperometric biosensor that Tucci et al. [24] used to detect herbi-
Potentiometric biosensors in terms of response time, energy range, and cides. A diagrammatic representation of the experimental working is
sensitivity. Amperometric biosensors work by analysing the sensor's own shown in Fig. 5.
current output to draw conclusions. It is possible to gauge the ampero- Biophotovoltaic systems, which gather light energy without using an
metric biosensor's sensitivity by comparing the currents measured at organic feedstock, have previously explored the use of photocurrent
various analyte concentrations. Two electrodes were used in this type of produced by living cells. Still, this idea has never before been applied to
biosensor, one to provide voltage and the other to measure the current. biosensing. The biosensor was constructed using a simple and inexpen-
Glucose sensing was the first step in the evolution of biosensors, and the sive preparation: live cells were encased in an alginate matrix and linked
majority of glucose sensing occurs through an enzyme's catalytic reaction to a carbon-felt electrode. Alginate is a biopolymer extracted from
with glucose oxidase [22]. Changing the temperature or the pH can alter seaweed that can encase cells, medicines, and proteins [25–27]. Because
the sensitivity of such glucose sensors. Amperometric glucose sensors use of the chemically inert aquatic environment inside the matrix [28],
cyclic voltammetry (CV) and impedance spectroscopy (EIS). An amper- immobilising microbial cells in alginate capsules allows for a high cell
ometric glucose sensor, in general, has a detection range of 1–15,000 density, improving the resilience of bacterial colonies [29]. Alginic acid

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M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

and accurate diagnosis. Ali et al. shed light on the latest developments in
biosensors that use amperometry and voltammetry to detect biomarkers
of AMI [34]. Biosensors based on voltammetry monitor AMI biomarkers
such as cardiac troponin (cTnI, cTnT) isoforms quickly and inexpen-
sively. cTnI and Fatty Acid Binding Protein (FABP) had LODs of 1.0 and
3.0 fg mL1, respectively. Zouari et al. [35] demonstrate how to make an
integrated biosensor for facile, quick, and sensitive miRNA detection via
direct hybridisation and label-free voltammetric detection. The biosensor
uses a disposable carbon electrode substrate with reduced graphene
oxide (rGO), and pyrene carboxylic acid (PCA), and 6-ferrocenylhexane-
thiol (Fc-SH) modified AuNPs. Fc-SH was employed as a chemical
messenger, and a synthetic amino terminal DNA capture probe was
covalently immobilised to PCA/rGO. Differential pulse voltammetry
measured the decrease in Fc's oxidation peak current after the DNA-RNA
duplex combination blocking electron transfer. Surface and electro-
chemical approaches were employed to assess the stepwise biosensor
fabrication, proving each component's involvement and miRNA de-
termination's reliability. The synthetic target of oncogene miRNA-21
enabled even a 5 fM breast cancer marker measurement with good sep-
aration of single-base mismatched variants in a single 30-min incubation
Fig. 5. Schematic representation of Anabaena variabilis cyanobacterial usage in phase.
herbicide detection. Image reproduced with permission from reference (24). A loss of appetite and exhaustion are just two of the many side effects
Copyright 2019, Elsevier. of the cancer drug Temodal [36]. Since determining Temodal in patients
receiving treatment is important, a DNA-based sensor for this purpose
is dissolved in water, and calcium ions are added to the mixture during was fabricated. A double-stranded Deoxyribonucleic acid (ds-DNA) and
the encapsulation process. Bacterial cells are trapped in insoluble calcium gold nanoparticles (Au-NPs) were placed on a graphite pencil electrode
alginate, which is produced when dissolved alginic acid reacts with Ca2þ. (ds-DNA/Au-NPs/PGE) to accomplish this. With and without 12.0 M
Meanwhile, the calcium alginate layer's high porosity allows for the rapid Temodal, the ds-DNA/Au-NPs/PGE differential pulse voltammetric
diffusion of macromolecules and nutrients [28]. Diuron and atrazine, two signal was collected (at the respective pulse height and width values of
common herbicides, were selected as substances to look out for. It was 50 mV and 5 ms). Guanine oxidation in dsDNA resulted in a 7.69 A
demonstrated that the biosensor's photocurrent, produced by the oxida- current signal at 781 mV. Temodal's presence in the ds-DNA structure
tion of water, is dose-dependent. There is a decrease in current strength was confirmed by a decrease in the oxidation current to 4.93 A and an
proportional to the herbicide concentration. The biosensor's LOD was increase in the oxidation potential of the peak to 800 mV when the
calculated to be 0.56 mM, and its sensitivity to atrazine was calculated to concentration of Temodal was 12.0 M. The linear response to concen-
be 24.6 m A mM1 cm2. Thanks to their unique characteristics, a wide tration Temodal was observed between 5.0 nM and 45.0 μM, and the
variety of nanomaterials have assisted significantly in biosensor devel- detection limit was found to be 1.0 nM. The reaction was traced back to
opment. ZnO nanorods and nanotubes are being used for the detection of Temodal's intercalation with guanine species in DNA. By adding an Au
urea [30] and cholesterol [31]. Cai et al. described a gold and poly- nanoparticle to the modified PGE's surface, the active surface area is
thionine electrode-based electrochemical immunosensor. An excellent increased by more than 5.1 times, which in turn helps to increase the
LOD of 2.2 pg/mL was achieved using the electrochemical immunosensor voltammetric analysis's detection limit.
for the analytes of interest [32].
2.3. Impedimetric

2.2. Voltammetric Impedimetric sensors are fabricated by immobilising bio-recognition

elements on an electrode surface. By generating an electrical impedance
Voltammetry is an electro-analytical method that measures current output whose strength is proportional to the analyte's activity, it enables
after varying voltage. Different techniques; such as Differential Pulse measurements and monitoring of that activity [23]. Electrochemical
voltammetry (DPV), Cyclic Voltammetry (CV), Linear Sweep Voltam- Impedance Spectroscopy is the Most Valuable Method (EIS). Electro-
metry (LSV), Square Wave Voltammetry (SWV) etc., are used in vol- chemical impedance spectroscopy (EIS) is a straightforward method for
tammetry. For their low cost, great selectivity, and high sensitivity, determining bulk electrode properties and interface processes. Nyquist
voltammetric methods are frequently utilised in biosensing systems. plots (semi-circular for the electron transfer process and a straight line for
Celiac disease is an autoimmune disorder triggered by ingesting diffusion) and Bode plots (logarithm of phase versus impedance) are both
gluten. There is a correlation between mucosal damage and tissue frequency functions.
transglutaminase IgA, endomysial IgA, and reticulin IgA. These anti- As their name implies, room-temperature ionic liquids (RTILs) are a
bodies are the most sensitive and specific for confirming Celiac disease. class of ionic solvents that remain liquid even when cooled to low tem-
Gupta et al. fabricated an early ultrasensitive detector for Celiac disease peratures [37]. Both the cation and anion are molten salts or oxides. It
based on transglutaminase recently [33]. DPV and CV techniques were was determined that ILs with a melting temperature below 100  C
used for the detection of anti-tTG antibodies, and even a 20–50 fgmL1 qualified these solvents. Because they are made up of ions of equal in-
range of analytes was successfully detected. The maximum current in the verse polarity, like a zwitter ionic assembly, these solvents are desirable
CV and DPV decreases with the rising of the anti-tTG serum concentra- because they are neutral. Most RTILs have a higher density and lower
tions. The redox current dropped because more antigen-antibody com- vapour pressure than water. RTIL molecules are made up of a big
plexes formed on the working electrode surface. The system used an non-symmetric organic cation having high electropositive and a modest
embedded MWCNT with Gold NP/GQD/PAMAM design platform. to relatively sized organic or inorganic anion having high electronega-
{NP-nanoparticle, GQD-Graphene Quantum Dot, PAMAM-poly (amido- tivity. Although they have high ionic and non-covalent preferences,
amine), MWCNT-Multi Walled Carbon NanoTube}. Acute myocardial making them chemically stable and non-conductive, the inclusion of a
infarction (AMI) patients' survival rates greatly depend on their timely hefty molecular group on both the cationic or anionic sides prevents

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M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

them from forming typical ionic connections. In light of these charac-

teristics, RTILs have been the subject of extensive study for their potential
use in a variety of biological processes, such as the synthesis of enzymes,
amino acids, peptides, etc. Most recently, Ventura et al. [37] presented a
novel strategy for formulating ionic liquids from biomass entities for the
liquid extraction of protein and DNA. Sayali et al. [38] demonstrated the
utility of RTIL as a reinforcing agent for grab probes in the development
of a diagnostics platform. Researchers looked at using a polyamide
membrane equipped with nanopores functionalised with antibodies to
identify IL-6 and cortisol present in human sweat. After optimising
throughout these time ranges, the LODs were 0.2 pg/mL for 1 day,
2 pg/mL for 1–2 days, and 5 pg/mL for 2–4 days [38].
A novel innovative four-electrode impedimetric biosensors were
created using photolithography to investigate tamoxifen's cytotoxicity on
cervical cancer cell lines by Pradhan et al. recently [39]. The novel four
electrode setup has an additional electrode to reduce noise for precise
impedance readings. Typically, the working electrode receives the
alternating current (AC) and sends it to counter electrode, while Refer-
ence electrode maintains a steady potential and the fourth standard
electrode is physically isolated from working electrode. Electric Cell -
Substrate Impedance Sensing (ECIS) was used to measure the impedance
of cells from 100 Hz to 1 MHz. Impedimetric biosensors show that
tamoxifen dose-dependently reduced the quantity of HeLa cells on elec-
trode surfaces. The cytotoxicity of tamoxifen was then determined by
comparing the impedance values obtained from the biosensors to those
obtained from more traditional methods such as the 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), the live-dead
cell test, and flow cytometric analysis. The toxic effects of tamoxifen to
HeLa cells can be measured both with conventional methods and by
using an impedimetric assay. ECIS can be used to estimate the cytotox-
icity of novel drugs because its IC50 values are comparable to those
obtained using the MTT assay. As frequency increases, there was a cor- Fig. 6. The electrical responses of a bendable strain sensor to various stimuli.
responding decrease in impedance. It is common knowledge that the An optical image of a strain sensor attached to the palm of a hand (A) and an
current moves through the cell at higher frequencies rather than the expanded optical image of the strain sensor (B) in action during deformation.
extra-cellular regions, as is the case at lower frequencies. In contrast, the Strain sensor electrical signals (ΔR/R0) (B) as a function of finger bending an-
value of the phase angle increases by as much as 1 MHz. Higher drug gles. (C) An optical image and electrical schematic of a strain sensor worn on the
concentrations result in greater drug-cell interactions, which in turn wrist to measure a person's pulse rate. Corresponding Electrical (V) signals from
causes cell death and a decrease in impedance, as seen in the observed the strain sensor during the pulse rate test in (D). Walking motion detection
optical image (E). (F) Strain sensor electrical signals (ΔR/R0) during the walking
differences between the control and drug-treated samples' curves. Lower
motion test. Image reprinted with permission, from Ref. [42].
than 10% Relative Standard Deviations (RSDs) were recorded for both
control and treated samples, indicating improved reproducibility in the
produced ECIS devices. reliably separated from the DA signal at 1 M or above using the DA-Ap
nanofilter-coated Gold gate FET. That is to say, while DA is blocked
from approaching the gold gate surface by the DA-Ap nanofilter, which
2.4. Potentiometric
forms compounds with counter ions to thwart the biomolecular charges
from making contact with the gold gate surface, L-DOPA is able to pass
In order for a biosensor of potentiometric nature to function, there
through the filter and electrochemically react with the gold gate surface
must be a difference in potential between the working electrode and the
to produce an electrical signal.
reference electrode. Unlike the amperometric biosensor, this technique
Glucose may now be detected in whole, unadulterated blood without
does not deplete the measured species. By gauging its performance
the need for any sort of sample pretreatment, thanks to a breakthrough
against a reference electrode, one can expect a linear relationship be-
technology developed recently that manages the mixed potential. Shift-
tween the analyte concentration and its response. Potentiometric bio-
ing the ionomer changes the quantitative linear range without affecting
sensors have the upper hand because of their sensitivity and selectivity
the sensitivity. Nafion-like polyelectrolyte Aquivion is employed to keep
when paired with a reliable reference electrode. These sensors are simple
the platinised paper electrode at a mixed potential, entrap the enzyme,
to manufacture, and they retain their performance even when their size is
and reduce negative charge interference [41]. Wong et al. created a
drastically reduced. An additional advantage of this biosensor is that the
flexible strain sensor based on ultrathin Au for using in body motion
signal is a potential rather than a current or impedance, and the bio-
capture and human-machine interactions [42]. It may be securely fixed
logical constituent utilised is an integral part of the receptor [23].
on the dermal surface for body motion capture thanks to its tiny di-
Himori et al. presented the idea of a nanofilter interface based on
mensions (5 mm x 2 mm x 6.2 mm ), skin integrability, excellent stability,
aptamers (Aps) for reducing background noise caused by contaminants in
and stretchability. Under 20% strain, it is capable of being stretched for
biological samples [40]. They demonstrated that l-3, 4-dihydroxypheny-
150 cycles at 1 Hz, and its electrical signal output, ΔR/R0, may increase
lalanine (L- DOPA) might be electrically differentiated from dopamine
to 0.76% and 0.61% in the transverse direction, respectively. The strain
(DA) by fabricating a DA-Ap nano filter-coated Gold gate field-effect
sensor for measuring heart rate and gait can be securely attached to the
transistor (FET). The proposed nanofilter interface is based on a DA-Ap
epidermal surface using a liquid bandage as the encapsulation. Addi-
layer that is anchored at an aryl-diazonium-based anchor monolayer
tionally, strain sensors are used in the robotic hand control, allowing for a
and is transplanted onto the Gold gate electrode by the techniques of
near-perfect recreation of human finger movements. Fig. 6 depicts the
diazonium chemistry. Therefore, the L-DOPA electrical impulses may be

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M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

electrical responses of the strain sensor to various stimuli. variables can be developed using AI and ML algorithms which can be
The results of Mishra et al. [43] describe a flexible, skin-worn used in cancer diagnosis and treatment.
biosensor that responds rapidly and precisely toward diisopropyl fluo- Although AI-assisted gene expression has proven to be an effective
rophosphate (DFP) in the fluid phase, making it suitable for monitoring approach, it has the disadvantage of having a small sample size. A crucial
G-type nerve agent simulants. While DFP is being hydrolysed by (OPH) requirement for linking people with IoMT devices is the unidirectional or
organophosphate hydrolase, the proton release can be detected by the bidirectional connectivity of technologies. The bulk electronics used for
epidermal biosensor patch. Using a wireless conformal electrical inter- sine wave conditioning are replaced by the analogue front end (AFEs).
face, sensing electrodes inscribed on temporary tattoo sheets can collect However, the operating room, intensive care units, and other areas with
data. The tattoo sensor can tolerate mechanical strain without impairing many gadgets are where Bluetooth data transmission is most appropriate.
analytical performance, along with a large dynamic range, fast response, A greater level of connectivity is provided by IoMT devices that are Wi-Fi
strong selectivity (even against organophosphate pesticides), and easy connected to the gateways. Bulk electronic components can be
implementation. By measuring the pH shift caused by the stoichiometric substituted with a chipset AFE; however, when the detector device is in
generation of hydrogen ions during the enzymatic hydrolysis of diiso- motion, internet signal connectivity may be lost, which could result in the
propyl fluorophosphate by organophosphate hydrolase enzyme, it is loss of crucial data and a delay in providing healthcare to a patient.
possible to identify the equivalent hazardous substrate. Nevertheless, a single AFE can frequently be applied to only one type
The novel potentiometric epidermal tattoo sensor's response was of biodetection. The issue of confidentiality and security exists even with
examined using standard McIlvaine's buffer solution before the custom- portable mobile apps that can manage health monitoring, sample col-
ised ‘skin e-tattoo’ biosensor (OPH/PANI) was tested for nerve agent lecting, data processing, etc. More advanced AI and Machine learning
simulant [43]. Polyaniline's key benefit is its conductivity, and pH algorithms are required for processing massive data because doing so
sensing capabilities via the shift in ionic strength between its emeraldine risks system failure [45].
salt and base forms. Liquid-phase DFP detection using OPH-modified Electronics, software, and communication advancements in the wake
tattoo biosensors was tested by monitoring the pH shift caused by the of the data-driven epoch have allowed for more precise and direct
biocatalytic hydrolysis of a dummy nerve agent. By varying the OPH measurements of solid waste output [46]. This has improved under-
loading (20–60 ng) and tracking the reaction to 80 mM DFP in the liquid standing and perception of the dire problems brought on by the surge in
phase, OPH loading at 60 ng provided the most optimal and steady trash output caused by the rapid expansion of the human population and
response. The stability of the sensor is crucial for an excellent wearable also by the technological advances and the urbanisation of cities. Ac-
sensor. By monitoring the change in potentiometric behaviour of a group cording to the EPA,-Environmental Protection Agency, solid waste in-
of freshly created tattoo sensors over time, the OP tattoo sensors' storage cludes anything that has been thrown away or used once and then
stability was assessed and showed sensor stability for up to 5 days. Good discarded, as well as sludge from water treatment or air pollution man-
stability is a result of a meticulous and thoughtful sensor setup. A tattoo agement facilities and the term “solid waste management” (SWM) is used
sensor was designed and tested for detection of DFP in the untreated lake to refer to the procedure of collecting, discarding, and managing
and pond water samples or various concentrations to evaluate its po- non-useable solid items; which is a pressing issue nowadays due to the
tential utility in the liquid phase, which showed a positive potentiometric fast digital development. When SWM is implemented poorly, it could
response to the nerve agent simulant, with clearly delineated changes in cause the degradation of natural resources and deterioration of living
the potential signal upon rising the DFP concentration. The team also standards and the environment, and it can foster the growth and spread
used gases like ethanol, acetone, ammonia, methanol, hydrogen of vector-borne diseases [46]. Industrial waste water that has undergone
peroxide, DNT, formaldehyde, isopropyl alcohol, toluene etc., for the anaerobic digestion is biodegradable and thus efficient and environ-
extra selectivity control study to investigate the DFP reaction to them. mentally safe. Alongside the skyrocketing growth of the sensing business,
The findings demonstrate that OP detection is not affected by the pres- several cutting-edge technologies are being developed to hasten the AD
ence of other non-target gases indicating a better specificity for the of waste water and solid waste management [47]. Microalgae have
wearable tattoo sensor. earned a reputation for resilience and longevity thanks to their capacity
to thrive in challenging settings [48]. Microalgae cultivation is viewed as
3. Future scopes and challenges of embedded biosensors with IoT environmentally friendly because of its low energy use and minimal
and AI and pandemic evolution of electrochemical biosensors waste output [49]. Their high sensitivity to metabolic activity changes
after toxin exposure makes them useful in biosensors. In a biosensor,
Biosensors that can be used at the point of care (POC) are often alterations in microalgal metabolic activities are translated into elec-
inexpensive and straightforward to use. Point-of-care real-time and vir- tronic or optical signals. Natural coagulants from the plant, animal, and
tual remote healthcare monitoring via biosensor devices may lead to microbial sources used in the sensor industry are an example to strive for
better patient treatment. Biosensors have been the subject of extensive in creating a healthy, sustainable, and environmentally friendly living
study over the recent decades due to their potential to detect patterns of space in the 21st century [48,50].
bioindicators and offer information on their presence in biological sam- The COVID-19 epidemic of 2020 was the worst public health emer-
ples, allowing for accurate diagnosis. The sorts of biosensors employed in gency the globe had seen in a century. The coronavirus is a positive-
future point-of-care technologies will be constrained by factors like the strand RNA virus with a large genome that belongs to the Nidovirales
need for fast label-free detection, miniaturised sensor size, and porta- group [51]. Worldwide, severe health and economic disasters loomed.
bility [44]. Artificial Intelligence (AI) is a cutting-edge, computer-- Because of its rapid contagiousness and severe effects, the entire world
science-based strategy for making machines clever and effective at jobs collaborated to create rapid detection kits and novel, powerful antiviral
that traditionally have required human intelligence [45]. medicines [51]. MicroRNAs (miRNAs) are a class of small, endogenous,
The most popular form of artificial intelligence (AI) for pattern non-coding, single-stranded RNAs that regulate gene expression by
recognition is called machine learning (ML). The review study by Man- binding to and silencing complementary sequences in the genomes of
ickam et al. analyses the benefits and drawbacks of several AI algorithms their intended targets [52]. The majority of RNAs degrade under the
in the context of the healthcare sector. AI also assists in gathering and kinds of harsh conditions that are commonplace, yet some strands are
processing a variety of data, including dangers, anatomical/physiological remarkably stable. Using logistic regression, we learned that detecting
details, genetic information, disease histories, and patient economics. three microRNAs, miR-423–5p, miR-23a-3p, and miR-195–5p allowed us
This data is used to better forecast procedures, even those involving heart to detect COVID-19 in its earliest stages [53]. Improvements in
or eye surgeries [45]. Additionally, prediction models for evaluating SARS-CoV-2 virus identification can be attributed in part to the POC
treatment response, prognosis, lymph node metastases, and other technique that incorporates an embedded AI-IoMT biosensor. The

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M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

Fig. 7. Schematic illustration of the proposed mobile phone method-assisted coronavirus detection. Reprinted with permission from Ref. [56].

following is a brief overview of some major discoveries that have 4. Recent significant biosensor discoveries
contributed to the advancement of technology.
Controlling the spread of pandemic diseases like COVID-19 requires IoT sensors of the future will be able to pick up on stimuli and
rapid testing that is nevertheless precise and sensitive. Some of the transmit signals wirelessly to recorders and analysers that have been
detection methods are slow and inconvenient to use at the POC (point of integrated with the IoTs. It might significantly reduce the time, human
care) level, despite being very sensitive and able to differentiate between resources, and resources required by conventional public management
different strains. In contrast, rapid tests necessitate a high titer and lack systems. Incorporating Internet of Things (IoT) sensors into COVID-19
the sensitivity to differentiate between strains [54]. An analyte-specific diagnostics, for instance, paves the way for the creation of a “hospital
detection of whole virions and separated proteins in microliter volumes on a chip” that can be used to diagnose SARS-CoV-2 even in the most
of physiological fluids can be accomplished in a matter of minutes using a inaccessible areas with access to modern medical care [59].
nanoscale, 3D molecular imprint-based potentiometric detection tech- To train computers to act like humans, one can use a machine learning
nique presented by Lee and colleagues [54]. A nanoscale inverse inter- (ML) technique called deep learning (DL). For a difficult task, DL relies on
face pattern of analytes formed by a self-assembled monolayer can be neural networks (NN), which need a lot of processing power. Advances in
used as a detection substrate to indicate the presence of an analyte by processing power and data analytics, however, have given DL algorithms
monitoring the change in open-circuit voltage as a function of time. The the ability to observe, learn, and respond to challenging situations.
sensor could identify H1N1 and H3N2 influenza-A virions, SARS-CoV-2 Depending on the task at hand, the DL algorithm can use supervised
and MERS-CoV spike proteins, and other respiratory viruses in human learning, unsupervised learning, or reinforcement learning (45). The best
saliva with 200 PFU/mL for virions and 100 pg/mL for spike proteins. real-world example of how AI may be put to use is in email provider
Favipiravir (FAV) is a treatment option for people infected with the services; there, it is used to filter out junk while keeping important
coronavirus, and it has been given the green light by the World Health messages in one place, and its accuracy increases as more data is added.
Organization (WHO). To accurately measure FAV, Mona et al. [55] By properly predicting the structure of a protein from its amino acid
created the first simple electrochemical, highly sensitive sensor sequence, the DL model “Alphafold” in 2020 solved an issue that had
employing MnO2-rGO nanocomposite. At pH 7.0, the linear response of persisted over half a century [60–62].
the current to changes in FAV concentration was determined to be be- One further subfield of artificial intelligence (AI) that must be fully
tween 1.0  10 -8 and 5.5  10-5 M under optimum experimental condi- integrated for practical use is natural language processing (NLP). Using
tions, with a LOD of 0.11 μM and a quantification limit of 0.33 μM. NLP, computers and machines can interpret, analyse, alter, and even
Pranjal Chandra [56] proposed a mobile phone-assisted easy and create human language [63]. It accepts either typed or spoken material as
user-friendly COVID -19 detection. Fig. 7 depicts the proposed theory for input. They can perform more complicated jobs if, NLP and ML algo-
label-free detection. rithms are used together. Virtual assistants are increasingly popular, with
Kodru et al. recently published a review on electrochemical sensors popular examples being Google Assist, Siri, and Alexa. Clinical notes have
used/developed during the COVID scenario [57]. An ultrasensitive recently been processed into a machine-readable format using NLP
electrochemical detection method for SARS-CoV-2 RNA was disclosed by techniques during COVID-19, which has helped bring attention to the
Zhao et al. [58] using calixarene-functionalised graphene oxide. The clinical history and current medical situation, subjective assessment data,
super sandwich-based recognition mechanism used in the research was and counsel given to them [64].
shown accurate by detecting viral RNA in vivo with an electrochemical Continuous glucose monitoring without needles or blood draws is
smartphone. The biosensor showed excellent selectivity and specificity in possible with the use of sweat sensors. Hima et al. aims to provide a
both in vitro and in vivo experiments. To date, 88 RNA isolates from 25 concise overview of the most recent advancements in non-invasive
confirmed SARS-CoV-2 patients and eight (8) patients in recovery have continuous glucose monitoring utilising sweat sensors based on several
been identified by the biosensor. Its sensitivity (56.5%) and specificity methodologies [65]. Various detection technologies, the sensitivity of
(7.7%) were also higher than those reported by RT-qPCR. The limit of their glucose detection, and their economic feasibility are discussed,
detection in clinical samples was set at 200 copies/mL. Furthermore, only along with the difficulties associated with sweat collection, sweat sample
two samples of SARS-CoV-2 (10 μL) were needed per experiment. deterioration, and individual variance in sweat amount [65].

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M.S. Sumitha, T.S. Xavier Hybrid Advances 2 (2023) 100023

Table 1 have begun to play a key part in artificial intelligence-assisted biosensor

Recent revolutionary developments in the biosensor field. for patient data processing, storage, sharing, user interface, and inter-
Sl.No Author Significant Remarks Reference Year connection with the cloud [76,77].
Table 1 below briefly summarises a few of the most significant
1 Yang et al.
Hospital –on- a chip system [78] 2021

MXene nanosheets breakthroughs in electrochemical biosensor technology in recent years.

Multifunctional microneedles
2 Chao et al.
e-skin [79] 2021 5. Conclusions

Titanium-based MXene/protein
nanocomposites

Motion sensor In order to analyse a biological response, biosensors turn it into an

Pressure/strain variation electrical signal. An exemplary biosensor should be sensitive just to its
detection target, be reusable after being used once, and be stable over a wide range
3 Lee et al.
1D Graphene nanoribbons/2D [80] 2021 of temperatures and pH levels. There has been a meteoric rise in the
MXene

Pressure sensor
number of articles devoted to the topic of electrochemical biosensors

Various sitting positions were over the past few decades. Incorporating nanomaterials into biosensors
trained through machine language has many benefits, including enhanced electrical, mechanical, chemical,
4 Kujawska
Graphene based [81] 2021 optical, and magnetic capabilities resulting in fast and easy electro-
et al.
Detection of dopamine
chemical detection. The health industry and even the economy have

Wearable skin-compatible device

Early detection Parkinson's disease altered as a result of the development of AI and IoT-assisted nano-
5 Qureshi
Pancreatic ductal adenocarcinoma [82] 2022 biosensors. The years of pandemics we've lived through have taught us
et al.
AI-assisted the need to work together to combat dangerous threats, as well as the role
6 Wanders
Interval breast cancer screening [83] 2022 that scientific research into biosensors plays in ensuring humanity's
et al.
Suitable for those who show even
negative mammogram
survival.

Uses Neural density network
Acknowledgement

Challenges in using sweat as the medium for glucose sensing We thankfully acknowledge DST-FIST, The Central Advanced
(65). Instrumentation Centre (CAIC), Government College for Women, Thir-
uvananthapuram, Kerala Government project “Performance Linked
➢ Skin is more prone to contamination which may affect the signal Encouragement for Academic Studies (PLEAS), and the University of
quality Kerala.
➢ It will be difficult for the microfluidic system to acquire a sufficient
quantity of the sweat sample due to the dermal blood vessels' role in References
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